Discussion

Chapter 3: Results and Discussions

3.4 Discussion

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Thr7 residues retains a hydrogen bond with His505 for around 50% of the simulation period (Figure 11G).

Unexpectedly, the terminal phenylalanine of camel Hem7 failed to secure considerable contact at the ACE2 active but instead forms a consistent

- interaction with Trp649 and a hydrogen bond with Ala348 as illustrated in Figure 11H. Rather, two active site contacts were exhibited by Trp6 via hydrogen bonding with Glu145 equivalent to Glu162 of ACE1 and a -

 contact with His345 equivalent to His353 of ACE1, though these are not considerably high. Trp6 makes strong hydrophobic interaction with Pro346 and to a lesser extent with Tyr515 (Figure 12H).

In terms of structural activity of ACE1 inhibitors, it has been reported that the ACE1 inhibitory action of a peptide relies on the presence of particular residues at the two termini (Silvestre et al., 2012); mostly a hydrophobic N-terminal segment carrying particularly leucine, isoleucine, valine and glycine (Akif et al., 2011), and aromatic residues such as tyrosine, proline, tryptophan, and phenylalanine and positively charged amino acids particularly lysine and arginine at the C-terminal end. These preferences favor higher ACE1 inhibitory action (Silvestre et al., 2012).

The N-terminal segment (LVV) of both ACE1 and ACE2 featured no active site interactions but instead made polar contacts and hydrophobic interactions with residues outside the active site, with the exception of Val3 of camel LVVHem7 which made hydrophobic interaction with Pro346 at the ACE2 active site as shown in Figure 12D. Though the N-terminal segment of the peptide showed no direct active site engagement except in only one of the sixteen complexes, ACE1 inhibition is observed to increase with their presence. This implies that the LVV segment contributes to stable binding at the active site as previously established (Liu et al., 2014; Kohmura et al., 1989; Moayedi et al., 2017). This is evidently supported by the two-fold increase in IC50 of VVHem6 (12.687 μM) relative to LVVHem6 (6.448 μM) (Table 3).

As illustrated in Figure 9, all polar active site contacts made with ACE1 are held by the C-terminal segment of the hemorphin peptides, namely positions seven to ten (Thr7, Arg/Gln8, Arg9, Phe10) while hydrophobic interactions at the ACE1 catalytic pocket are mediated mostly by Trp6 and to a much lesser extent Tyr4 and Pro5 (Figure 10).

As for ACE2 active site interactions, the polar nature are held mostly by residues ranging from position six to ten (Trp6, Thr7, Arg/Gln8, Arg9, Phe10) as shown in Figure 11; while the hydrophobic ones are mediated

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primarily by Trp6, with Tyr4 and Tyr5 securing a few interactions as illustrated in Figure 12. Uniquely, Phe10 of LVVHem7 features two hydrophobic contacts at the catalytic site of both ACE1 and ACE2 (Figures 11F and 12F).

Altogether, the ACE1-hemorphin interaction interface is similar to that of ACE1 with its clinically relevant inhibitors RXPA380, lisinopril, captopril, and enalaprilat which altogether interact with Glu162, His353, Ala354, His383, Glu384, His387, Glu411, Lys511, His513, Tyr520, and Tyr523 of ACE1. As listed in Table 4, all these interactions were observed in the ACE1-hemorphin complexes, with the exception of His383. Other direct interactions made by the commonly used ACE1 drugs outside the catalytic pocket were observed with Tyr62, Asn66, Lys118, Glu123, Arg124, Trp220, Trp357, Val380, Phe391, Ser355, Glu376, Val379, Val380, Glu403, His410, Phe457, Phe512, Val518, and Arg522 (Akif et al., 2011). These were all observed in the molecular docking of the hemorphin peptides with ACE1, excluding the residue Glu376 (Table 4). As shown by the MD simulations, all the common interactions between ACE1 inhibitors and hemorphin peptides are significantly preserved except those with Glu384 and His387 (Figures 9 and 10). The hemorphin-Glu384 interaction was observed only once; with LVVHem5 (Figure 11G), the hemorphin reporting the second highest IC50. Similarly, Figure 11H shows that the His387-hemorphin contact was observed in a single occurrence with camel Hem7, the peptide exhibiting the highest IC50. Additionally, hemorphin interaction with ACE1 features interactions not exhibited by ACE1 blockers.

For instance, all the hemorphins exhibited an interaction with Ala356 close to the Ala354 at the active site of ACE1 as reported in Table 4, an interaction that was absent in the interactions of ACE1 inhibitors. Furthermore, the water mediated hydrogen bond contact exhibited by ACE1 inhibitors

lisinopril and RXPA380 with Asp415 (Akif et al., 2011) were replaced by direct hydrogen bond and salt-bridge contacts in the case of hemorphin interaction with ACE1 (Table 4).

The ACE2 inhibitor MLN-4760 secures direct hydrogen bonding with Arg273, His345, His505, Thr371, and Pro346 of ACE2. It also makes close contact with Glu145, Asn149, Phe274, Lys363, Asp368, Met360; and the disulfide linkage of both Cys344 and Cys361 which create a hydrophobic space at the ACE2 catalytic site (Towler et al., 2004; Lubbe et al., 2020). Of these residual interactions, all but that of Asp368 are observed in the association of hemorphins with ACE2 as reported in Table 5; and all these contacts are sustained with the exception of contacts with Thr371, Lys363, Met360, and Cys361 as illustrated in Figures 11 and 12 (Towler et al., 2004).

A positive association between the placement of arginine near the C-terminus and higher ACE1 inhibition has been reported (Sun et al., 2017;

Ali et al., 2020a). Changes in peptide conformation due to the presence on an arginine promoting deeper placement of the N-terminal segment into the ACE1 catalytic pocket which has been established to be significant in improving ACE1 inhibition (Zhao et al., 1994; Sun et al., 2017; Kohmura et al., 1989. In ACE1, this occurrence was observed with LVVHem7 where the N-terminal (LVV) made four polar contacts, namely a salt-bridge with Asp121 and Glu123, and a hydrogen bond with Glu123 and Tyr135; with average contact length of 73% as shown in Figure 9F. On the other hand, camel LVVHem7 makes four polar interactions, namely two salt-bridge contacts and two hydrogen bond interactions with Asp121 and Glu123; with average interaction time of 86% as illustrated in Figure 9D. Similarly, as shown by Figure 9A, the N-terminal residues of camel LVVHem6 made two polar contacts with Glu123 and one with Arg522 of ACE1 while LVVHem6

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also maintains two polar contacts with Glu123 of ACE1 as depicted in Figure 9B, but with lesser strength relative to camel LVVHem6. This phenomenon was even more evident in ACE2, where the N-terminal segment of camel LVVHem6 secured seven polar contacts while LVVHem6 only exhibited two as depicted in Figures 11A and B, respectively.

Additionally, the N-terminal segment of camel LVVHem7 featured two sustained polar interactions with ACE2 while LVVHem7 exhibited a single one for less than 50% of the simulation length as illustrated in Figures 11D and F, respectively. Camel LVVHem6 and camel LVVHem7 also exhibited greater hydrophobicity with ACE1 and ACE2, respectively in the presence of arginine at the C-terminal segment (Figures 10A and 12D).

The introduction of additional interactions resulting from the substitution of glutamine to arginine in camel hemorphins was also observed with polar contacts of camel LVVHem5 and camel LVVHem7 with ACE1 relative to their non-camel counterparts as illustrated in Figure 9. The Arg8 of camel LVVHem5 exhibited overall strong salt-bridge contacts with Asp415, Asp453 and Lys511; and a hydrogen bond with Tyr523 while the Gln8 of LVVHem5 made a single H-bond with His353 of ACE1 for just nearly half the simulation length. Likewise, Arg8 of camel LVVHem7 was involved in a salt-bridge with Asp453 and a hydrogen bond with Asp415 while Gln of LVVHem7 made a single hydrogen bond with Glu411 of ACE1. There were similar observations in the case of ACE2. Camel LVVHem6 secured a salt-bridge interaction with Asp367 of ACE2 via its replaced arginine, while the Gln8 of LVVHem6 failed to mediate any significant interaction with ACE2 as shown in Figure 11A and 11B, respectively. Additionally, camel LVVHem5 maintained overall stronger polar interactions with Pro346, Glu375 and Arg514 of ACE2 via its C- terminal arginine relative to LVVHem5 with Pro346, Glu375, Tyr515, Arg518 via its C-terminal Gln8 as illustrated in Figure 11C and 11G,

respectively. Even more evident, Arg8 of camel LVVHem7 made interactions with Asp382 and Ala348 of ACE2 as shown in Figure 11D while Gln8 of LVVHem7 exhibited none (Figure 11F).

Unexpectedly, camel LVVHem7 (11.06 μM) reported a higher IC50

against ACE1 relative to camel LVVHem6 (5.16 μM), LVVHem6 (6.58 μM), and camel LVVHem5 (6.62 μM) as listed in Table 3. As the hemorphin reporting the lowest IC50, camel LVVHem6 docked at all three subsites of ACE1 forming four hydrogen bond interactions along with a salt-bridge and altogether three hydrophobic interactions at the S1 and S2 subsites as reported in Table 4. The MD simulations show that out of these eight interactions, six were retained, with two additional active site interactions, specifically polar contacts with both His353 and His513 (Figures 9A and 10A).

Camel LVVHem7 docked at two ACE1 subsites making only two hydrogen bond contacts and two hydrophobic interactions at the active site as reported in Table 4. The MD simulations showed that the C-terminal Phe10 of camel LVVHem7 retained only one significant hydrogen bond contact at the ACE1 active site, namely with Gln281 for 56% of the simulation length as illustrated in Figure 9F. It also exhibited polar contacts with Asn277 and Thr282 for an average of 64% interaction time along with four hydrophobic interactions with non-active site residues with an average interaction time of 80%. Another active site was held by Arg9 with Glu162 for 75% of the simulation length. Additionally, the Arg8 of camel LVVHem6 made two significant polar contacts, one of which was at the active site, while the equivalent arginine residue of camel LVVHem7 made two polar contacts with residues relatively distant from the catalytic region as shown in Figure 9A and D, respectively. Furthermore, the N-terminal segment (LVV) of camel LVVHem6 mediated a greater number of hydrophobic interactions with average contact strength of 88% relative to

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camel LVVHem7 which averaged to 67% as illustrated in Figure 10A and D, respectively. As for the persistence of hydrophobic interactions at the active site, Tyr4, Pro5 and Trp6 of camel LVVHem6 maintained strong contacts with Tyr523 as depicted in Figure 10A; while only Trp6 of camel LVVHem7 showed contact with Tyr523 as shown in Figure 10D. The above observations could potentially justify the almost two-fold IC50 of camel LVVHem7 (11.15 μM), relative to that of camel LVVHem6 (5.12 μM) (Table 3).

In comparison to LVVHem6 camel (5.12 μM), LVVHem6 (6.45 μM) and camel LVVHem5 (6.89 μM) exhibit marginally higher IC50. LVVHem6 docked at two of three subsites exhibiting four hydrogen bonds, three hydrophobic interactions, and a salt-bridge at the ACE1 catalytic region as listed in Table 4; while camel LVVHem5 docked at only one of the three ACE1 subsites, exhibiting a single hydrogen bond and two hydrophobic interactions at the active site. Interactions of the docked complexes of both LVVHem6 and camel LVVHem5 were retained during the MD simulations, and additional sustained active site contacts were formed as shown in Figures 9B and C, respectively. LVVHem6 sustained a total of seven polar contacts with average contact length of 89% and two hydrophobic contacts with average 82% interaction time at the ACE1 active site; while camel LVVHem5 retained three polar contacts with average contact length of 87% and a single hydrophobic contact with 84%

interaction time at the ACE1 active site (Figure 10C). Overall, camel LVVHem5 exhibited more consistent hydrophobic associations relative to LVVHem6 (Figure 10B). Another surprising observation was the comparable IC50 of VVHem6 (12.69 μM) and that of LVVHEM7 (13.07 μM) (Table 3). Though VVHem6 lacks an N-terminal leucine, its two valine residues mediated most of its ACE1 polar contacts with average contact time of 98% and also mediated mostly strong hydrophobic associations with 93%

contact time on average (Figures 9E and 10E). By contrast, the LVV segment of LVVHem7 mediated a greater number of hydrophobic interactions with a lesser average contact length of 82% (Figure 10F).

However, VVHem6 secured no hydrophobic contacts at the active site while LVVHem7 made two with Tyr520 and Tyr523 via its C-terminal Phe10 residue for the entirety of the simulation as shown in Figure 10E and F, and also featured more polar active site interactions at the ACE1 catalytic pocket (Figure 9F).

Contrasting the IC50 values of LVVHem6 (6.45 μM) and VVHem6 (12.69 μM) highlights the significance of the presence of the N-terminal leucine though the Leu1 residue of LVVHem6 fails to mediate any active site interactions (Table 3) .

The rather surprising report of camel Hem7 (23.57 μM) as the peptide exhibiting the highest IC50 with ACE1 among the top eight peptides correlates with the in silico results. The molecular docking results report that camel Hem7 exhibited no contact at the active site apart from a single hydrophobic interaction with Tyr523 at S1 (Table 4). Instead, it is the only peptide that interacted with His387 at the Zn-coordinating motif, through an aromatic - contact (Table 4). Similarly, the MD simulations revealed no polar active site interaction of camel Hem7 but reported two hydrophobic interactions with Ala354 and Tyr523 at the S1 subsite of ACE1, mediated by its Pro5 residue (Figure 10H). Its C-terminal Arg8 and Phe10 residues failed to exhibit considerable contact at the active region.

Since there was no compatible ACE2 kit for in vitro confirmatory assay, the analysis of ACE2 binding remain on the basis of in silico analysis with camel LVVHem7 exhibiting the top MM-GBSA binding energy of – 108.92 kcal/mol and LVVHem7 unexpectedly reporting the lowest of – 59.09 kcal/mol (Table 5).

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Guy et al. (2005) found the positive side chain of Arg273 to be key for substrate binding and that the Arg273Lys substitution eliminates enzyme action; as maintaining a positive charge at position 273 is insufficient for docking into the ACE2 active site. They also establish the relevance of both His345 and His505, but particularly the former, as their substitution drastically reduces enzyme activity.

Arg273 engages in a salt-bridge with the C-terminus of the ACE2 inhibitor, MLN-4760 (Dales et al., 2002) and peptide ligands (Guy et al., 2005). This was observed with four of the hemorphin peptides — LVVHem6, VVHem6, LVVHem5, and LVVHem7; LVVHem6 and camel LVVHem7 — which form a hydrogen bond with Arg273 as shown by the molecular docking analysis (Table 5).

The molecular docking results listed in Table 5 show that the peptides exhibiting the top-two MM-GBSA scores — Camel LVVHem7 (-108.92 kcal/mol) and camel Hem7 (-107.81 kcal/mol) — engage in hydrophobic interactions with Cys344 and Cys361, which represent one of the three conserved disulfide bridges between ACE1 and ACE2, the other two being Cys133–Cys141 and Cys530–Cys542 (Towler et al,. 2004).

Additionally, they share two common active site hydrogen bond interactions with Pro346 and Glu375. Camel LVVHem7 forms a third polar contact with Arg273 while LVVHem7 engages His345 at the ACE2 active region. They both interact with active site residues Pro346 and Tyr515 hydrophobically and share these two interactions with the remaining six peptides.

The Trp6 of camel LVVHem7 features  -stacking with His374 at the active and a hydrogen bond with Glu406 close to the zinc-binding motif while the same residue of camel Hem7 sustains two active site contacts, through -stacking with His345 and a hydrogen bond with Glu145. On the contrary, the LVVHem7 variant not only fails to engage the Cys344–Cys361 disulfide–bridge but reports the least MM-GBSA binding energy of -59.09

kcal/mol despite forming interactions at the active site with Arg273, His345, Pro346, Glu375, and Tyr515. However, the MD show that the only polar interactions of LVVHem7 with Arg273 are preserved through a salt-bridge and hydrogen bond contact via its C-terminal Phe10 throughout the simulation period as shown in (Figure 11F); an additional consistent salt- bridge with Glu145 at the active site was mediated by its Arg9 at the C- terminal segment. The C-terminal Phe10 further stabilizes the peptide through its hydrophobic associations with Pro346 and Tyr515, each with 99.5% contact length.

Though lacking an N-terminal leucine, VVHem6 (-102.25 kcal/mol) exhibits comparable MM-GBSA binding energy to LVVHem6 (-104.41 kcal/mol). The docking results reveal that it is the only peptide forming interaction with the critical His505; it forms additional interactions with two other critical residues —Arg273 and His345 — in addition to Pro346 and Tyr515 (Table 5). The MD simulations reports that of these, the interactions with Arg273, Pro346 and Tyr515 are retained through both a salt-bridge and hydrogen bond with Arg273, a hydrogen bond with Tyr515, and a hydrophobic contact with Pro346 (Figure 12E). These two peptides — VVHem6 and LVVHem6 — exhibit a slightly higher MM-GBSA relative to camel LVVHem6 (-99.50 kcal/mol) which unlike LVVHem6 and VVHem6 secures no contact with the three critical residues of ACE2, namely Arg273, His345, and His505 as per the molecular docking results (Table 5). However, the MD simulations reveal that Arg273 is significantly engaged via the C-terminal Arg9 of camel LVVHem6 through both a salt- bridge and hydrogen bonding (Figure 11A).

In ACE1, ligand binding at the S2 subsite is involved in the placement and orientation of peptide substrates for dipeptidyl carboxypeptidase cleavage. Furthermore, all published ACE1 native structures report a ligand being bound to this subsite (Natesh et al., 2003;

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Corradi et al., 2006; Watermeyer, 2006). By contrast, none of the ACE1 residues at the S2 subsite are conserved in ACE2 (ACE2:Arg273, Leu503 and Phe512; ACE1: Gln281, Lys511 and Tyr520, respectively) and these changes explain why ACE2 is a carboxypeptidase, cleaving only a single residue off the C-terminus of peptides (Lubbe et al., 2020).

Apart from stabilizing inhibitors and peptide ligand, the larger size of Arg273 (relative to ACE1 Gln281) promotes steric crowding at the probable S ’ subsite. This non-conserved residue in ACE2 practically eliminates the C S ’ and provides a justification for the functional selectivity in the peptidyl dipeptidase action of ACE1 and the carboxypeptidase action of ACE2. Such poorly conserved regions between the homologous enzymes also explains the inactivity of clinically used ACE1 inhibitors captopril lisinopril, and enalaprilat that dock at the S1 and S  subsites against C and underlies the molecular basis for inhibitor and substrate specificity; and cleavage activity. Additionally, non- conserved residues beyond the ACE2 catalytic pocket have been reported to possibly influence affinity of certain ACE inhibitors. For instance, Tyr510 (ACE1 Val518) and Thr347 (ACE1 Ser355) of ACE2 engage only small and medium sized side chains such as leucyl and prolyl, which is consistent with the known substrate preferences which imparts even greater specificity (Towler et al., 2004; Lubbe et al., 2020). All the tested hemorphin peptides formed hydrophobic interactions with Val518 of ACE1 and Tyr510 of ACE2 as listed in Table 4 and 5, respectively.

Despite the poorly conserved residues between the S1 subsite of the two ACE homologs, there exists a certain extent of similarity in terms of substrate preference for more extended sidechains or hydrophobic residues (Natesh et al., 2003; Corradi et al., 2006; Towler et al., 2004). Another dissimilarity is the additional chloride-binding site in ACE, while only one

was observed in ACE2 and this manifests in the chloride tolerance between the two homologous enzymes (Guy et al., 2005).

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